Sample Preparation Device and Methods of Use

ABSTRACT

A device for isolating DNA from a sample containing cells, including a cartridge having an entrance port and an exit port, a membrane disposed between the entrance port and the exit port, and a plurality of channels between the membrane and the exit port. Additionally, systems and methods for isolating DNA from a sample containing cells and also systems and methods for amplifying and isolating single-stranded DNA from a sample containing DNA.

CROSS-REFERENCE To RELATED APPLICATIONS

This U.S. patent application claims priority to U.S. ProvisionalApplication: 61/791,766 filed on Mar. 15, 2013, the disclosure of whichis considered part of the disclosure of this application and is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel devices, systems, and methods forisolating DNA from a sample containing cells.

BACKGROUND

Isolating DNA from a sample is important for many diagnosticapplications. DNA detection is utilized for diagnostic applications inmedicine, food safety, environmental testing, and forensic science.Isolated DNA may be analyzed using methods such as polymerase chainreaction (PCR), electron microscopy, Western blotting, or othertechniques. Isolating single-stranded DNA (ssDNA) is of particularimportance in diagnostic applications where detection is based onhybridization of the ssDNA with a DNA probe. In samples containing arelatively low number of cells, DNA amplification may also be desirableto produce a detectable level of DNA.

Current techniques for DNA isolation involve the use of a membrane orfilter to trap DNA after a cell is lysed to release DNA from the cell.This trapping is often followed by elution of the DNA from the membraneusing an elution buffer. Such methods can be time consuming, requiringmultiple washing and elution steps. In some cases, such methods mayresult in a low yield of DNA. In some cases, the DNA isolation systemsmay not be suitable for point-of-care use. Some methods may have asample output that is unsuitable for integration with functions such asDNA amplification and DNA detection assays. For example, if the sampleoutput is contaminated with non-DNA cellular material the efficiency ofdownstream functions is reduced. Other disadvantages in known methodsinclude a lack of support for the membrane. This may result in tearingof the membrane, wherein pieces of the membrane may contaminate thesample.

SUMMARY OF THE INVENTION

One aspect of the invention is a device for isolating DNA from a samplecontaining cells, comprising a cartridge having an entrance port and anexit port; a membrane disposed between the entrance port and the exitport; and a plurality of channels between the membrane and the exitport.

Another aspect of the invention is a method of isolating DNA from asample, comprising providing a sample containing cells; transferring thesample into a container (“cartridge”) having a membrane and a pluralityof channels disposed therein; lysing the cells to release their DNA andother (non-DNA) cellular material from the cell; and passing thereleased DNA through the membrane and through the channels.

Another aspect of the invention is a system for isolatingsingle-stranded DNA from a sample containing cells, comprising acontainer (“cartridge”) having an entrance port and an exit port; amembrane disposed within the container between the entrance port and theexit port; and a plurality of channels located between the membrane andthe exit port; and an amplification and separation system comprising anamplification container; a thermal cycler; and a magnetic substrate.

Another aspect of the invention is a method of isolating single-strandedDNA from a sample, comprising providing a sample containing cells;transferring the sample into a container (“cartridge”) having a membraneand a plurality of channels disposed therein; lysing the cells torelease the DNA and non-DNA cellular material from the cell; passing theDNA through the membrane and the channels; transferring the released DNAto an amplification container, amplifying the released DNA via apolymerase chain reaction (PCR) to produce double-stranded DNA andsingle-stranded DNA; and separating the double-stranded DNA from thesingle-stranded DNA by applying a magnetic field.

In some embodiments the PCR includes asymmetric PCR to producesingle-stranded DNA (ssDNA). In another aspect a magnetic substrate withbinding affinity for double-stranded DNA (dsDNA) is provided and amagnetic field is applied to the substrate to isolate the ssDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described, by way of example, with referenceto the accompanying drawings in which:

FIG. 1 presents a cross-sectional view of an embodiment of a device forisolating DNA from a sample containing cells.

FIG. 2 presents an outer perspective view of an embodiment of a devicefor isolating DNA from a sample containing cells.

FIG. 3 is a graph illustrating the theoretical production of DNA overtime for traditional PCR followed by asymmetric PCR (APCR).

FIG. 4 presents a cross-sectional view of an embodiment of anamplification container with a magnetic substrate.

FIG. 5 presents an embodiment of a system for isolating single-strandedDNA from a sample containing cells.

FIGS. 6A-D present an example protocol for carrying out an embodiment ofa method for isolating single-stranded DNA from a sample containingcells.

FIG. 7 presents an example of components used in the manufacture of anembodiment of a device for isolating DNA from a sample containing cells(e.g., a cartridge).

FIGS. 8A-C present an illustration of an embodiment of an top piece of acartridge (e.g., a top SPC).

FIGS. 9A-B present an illustration of an embodiment of a bottom piece ofa cartridge (e.g. a bottom SPC).

FIG. 10 presents an illustration of a graph of real impedance versus theimaginary impedance for an exemplary electrochemical detection assayusing isolated ssDNA.

DETAILED DESCRIPTION

As used herein, the following definitions shall apply unless otherwiseindicated.

I. Definitions

As used herein, “DNA” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single-stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). An “isolated” nucleic acid molecule is one thatis separated from other nucleic acid molecules that are present in thenatural source of the nucleic acid.

As used herein “ssDNA” refers to single-stranded DNA and “dsDNA” refersto double-stranded DNA.

As used herein, “isolating” refers to separating a first component fromother components and includes partial separation wherein the firstcomponent is not totally separated from the other components andimpurities of the other components remain. Isolating may be thought ofas concentrating the first component relative to other components.

As used herein, “cartridge” refers to a container.

As used herein, “channel” or refers to an opening defined by boundarywalls.

As used herein, “cellular homogenizer” refers to a mechanical devicecapable of shaking an object, for example the cartridge describedherein. For example, a cellular homogenizer may be capable of shaking anobject at a speed of up to about 5000 rpm.

As used herein, “PCR” refers to polymerase chain reaction. “AsymmetricPCR” (APCR) is a form of PCR that preferentially amplifies one strand ofa target dsDNA. It is to generate an ssDNA as product. Thermocycling iscarried out as in traditional PCR, but with APCR (as compared totraditional PCR) an excess of the primer for the strand targeted foramplification is used, or a limiting amount of one of the other primersis used or is absent. When the limiting primer becomes depleted,replication increases arithmetically through extension of the excessprimer.

As used herein, “thermal cycler” refers to an apparatus configured tomanipulate temperature. For example a thermal cycler may be configuredto manipulate the temperature of a liquid sample. A thermal cycler maybe configured to cycle a sample between various temperatures for adesired PCR protocol.

II. Devices and Systems

Cartridge Product

One aspect of the invention is a device for isolating DNA from a samplecontaining cells, comprising a container 10 having an entrance port 16and an exit port 18; a membrane 12 disposed between the entrance port 16and the exit port 18; and a plurality of channels 14 between themembrane 12 and the exit port 18.

In one embodiment of the present invention, the channels 14 are adjacentto the membrane 12.

In another embodiment, each channel 14 has a channel entrance 24, achannel exit 26, wherein the channel entrance 24 is adjacent to themembrane 12.

In a further embodiment, the channels 14 are substantially linearchannels with the channel entrance 24 adjacent to the membrane 12 andthe channel extending linearly from the channel entrance 24 to thechannel exit 26 in a direction substantially perpendicular to theadjacent membrane surface or in a direction generally towards the exitport 18 of the cartridge 10. In some embodiments the substantiallylinear channels are substantially parallel to one another. In someembodiments the channels 14 are curved or bent channels with the channelentrance 24 adjacent to the membrane 12 and the channel exit 26extending toward the exit port 18 of the cartridge 10 and to support themembrane 12.

In some embodiments the plurality of channels 14 are arranged in amatrix. For example the channels 14 form a grid-like matrix or thechannels 14 may form a matrix extending radially outward from a centerpoint. In some embodiments the channels 14 are directly adjacent to eachother or alternatively the channels 14 may be spaced apart from eachother. In some embodiments the matrix of channels 14 further comprises asolid barrier 30 between the entrances 24 of each channel to direct theflow through the channels.

In one embodiment, the device has from about 2 to about 1000 channels14, for example, from about 10 to about 100 channels 14.

In some embodiments the dimensions of the channels 14 are selected tooptimize the flow of DNA out of the cartridge 10. For example thechannels 14 have a dimension of their cross-section that results in aminimal number of DNA being held in the channels 14. For example, thechannels have at least one dimension that is from about 0.1 mm to about10 mm. For example the width of the cross section of the channels isfrom about 0.1 mm to about 10 mm and the length of the cross section ofthe channels is from about 0.1 mm to about 100 mm.

Without being bound by theory, it is believed that channels with toolarge of a dimension can result in DNA being held at the walls of thechannels without being carried by the flow of liquid through the channeland out of the cartridge. In some embodiments the channels 14 have atleast one dimension that is from about 0.1 mm to about 10 mm so that theamount of DNA held at the channel walls 28 is minimized. Without beingbound by theory, it is believed that channels with too small of adimension can result in DNA blocking flow through the channels or canresult in irregular flow patterns that keep DNA from being carried bythe flow of liquid through the channel. In one embodiment, the channels14 have at least one dimension that is from about 0.1 mm to about 10 mmso that the amount of DNA blocking the channel is minimized. In someembodiments the channels have at least one dimension that is from about0.1 mm to about 10 mm so that the amount of irregular flow patterns isminimized

In some embodiments each channel entrance 24 is substantiallyrectangular and has a length and a width. In some embodiments thechannel entrance 24 has a length of about 0.1 mm to about 100 mm and awidth of about 0.1 mm to about 10 mm.

Alternatively, each channel entrance 24 may be substantially circularand have a diameter. In some embodiments the channel entrance 24 has adiameter of about 0.1 mm to about 10 mm.

In some embodiments the channels 14 and/or channel entrances 24 form aconcentric pattern around a central axis of the cartridge. For exampleFIG. 9B shows concentric channels around a central axis of thecartridge.

In some embodiments the channels 14 have a substantially consistentcross section across their entire length. In some embodiments the crosssection varies across the length of the channel 14. For example thecross section at the channel entrance 24 has a smaller area than thecross section at the channel exit 26.

In some embodiments each of the channels 14 have substantially the samecross section or cross section profile as one another. In otherembodiments the cross section varies from one channel 14 to another.

In some embodiments the channels 14 have a depth of about 0.1 mm toabout 10 mm, for example about 0.5 mm.

In some embodiments the membrane 12 is made from a cellulose material.For example the membrane comprises cellulose nitrate or celluloseacetate 12. In some embodiments the membrane comprises a polycarbonatematerial 12. In some embodiments the membrane comprises a membranematerial known in the art for use in isolating DNA from cells.

In some embodiments the membrane 12 is a layered membrane. For examplethe membrane 12 has two or more layers, e.g., three or more layers.

In some embodiments the membrane 12 has pores. The pore size may beabout 10 nm to 1 micron. For example, the membrane 12 has a pore size offrom about 100 nm to about 300 nm, e.g., about 200 nm. The pore size isselected to maximize the yield of DNA passing through the membrane 12and to minimize the amount of cells and non-DNA cellular material thatcan pass through the membrane 12.

The membrane 12 may be configured to be permeable to DNA butsubstantially impermeable to cells. In some embodiments, the membrane 12is configured to be permeable to DNA but substantially impermeable to“non-DNA cellular material” (i.e., cellular debris), which includes, butis not limited to, material other than DNA released form a lysed cellsuch as proteins, cell walls, and organelles. The membrane 12 ispermeable to DNA when, for example, 50%, 60%, 70%, 80%, 90%, 95%, or 99%or more of the DNA released from the cells flows through the membrane.In some embodiments, membrane 12 is substantially impermeable to non-DNAcellular material when, for example 50%, 60%, 70%, 80%, 90%, 95%, or 99%or more of the non-DNA cellular material does not flow through themembrane. Similarly, membrane 12 is substantially impermeable to cellswhen, for example 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of thecells do not flow through the membrane.

In some embodiments the membrane 12 allows particulates smaller thanabout 100 nm to about 1000 nm to pass through but is impermeable toparticulates larger than about 100 nm to about 1000 nm. For example themembrane 12 allows particulates smaller than about 200 nm to passthrough but not particulates larger than about 200 nm. It may bepossible from some non-DNA cellular material having a size approximatelyequal to or smaller than DNA (“minor cellular debris”) to pass throughthe membrane 12, however such minor cellular debris has a minimal effecton downstream applications.

In some embodiments the device further comprises a plurality ofparticulate material 20, (e.g. beads), within the cartridge 10, locatedbetween the membrane 12 and the entrance port 16. In some embodimentsthe space located between the membrane 12 and the entrance port 16defines a lysis chamber 22.

In some embodiments the particulate material 20 is a plurality ofmicrobeads. The microbeads may be substantially spherical. In someembodiments the particulate material 20 comprises a ceramic material(e.g., glass) or a metal material (e.g. stainless steel). For example,the particulate material 20 is glass microbeads or stainless steelmicrobeads. In some embodiments the microbeads have a diameter of fromabout 10 micron to about 1000 microns, for example about 200 to 400microns.

In some embodiments the entrance port 16 and exit port 18 includestandard luer lock fittings 17 and 19.

The cartridge 10 may include an top piece, referred to as a top SPC(sample prep cartridge) 32 and a bottom piece, referred to as a bottomSPC 34. In some embodiments the cartridge is made of a plastic material,e.g. polypropylene.

In some embodiments the top SPC 32 and bottom SPC 34 are sealedtogether, e.g., the top SPC 32 and bottom SPC 34 are sonic weldedtogether. In this embodiment the membrane 12 is held in place by the topSPC 32 and bottom SPC 34. For example, the membrane 12 is locatedbetween the top SPC 32 and bottom SPC 34 and held in place when the topSPC 32 and bottom SPC 34 are sealed together. In some embodiments theedges of the membrane 12 are stretched into place at the sealed boundaryof the top SPC 32 and bottom SPC 34.

In some embodiments the channels 14 are disposed within the bottom SPC34. For example the channels 14 are defined as cavities within thebottom SPC 34 and the channels 14 allow for flow of DNA and liquidthrough the membrane 12 and out of the cartridge 10 through the exitport 18. In some embodiments the membrane is positioned across a channelmatrix, wherein the channel matrix is defined as a plurality of cavitiesin the bottom SPC 34.

In some embodiments the bottom SPC 34 and the channel matrix act as asupport for the membrane 12 while allowing for the flow of liquid out ofthe cartridge 10. For example, the membrane 12 is held in place by theseal of the top SPC 32 and the bottom SPC 34 and is supported by aninner surface of the bottom SPC 34 while still allowing for flow throughthe membrane 12 and out of the cartridge 10 due to flow path provided bythe channels 14. In some embodiments the support provided to themembrane 12 results in the membrane 12 remaining intact during use, forexample, during mechanical lysis. In some embodiments the membrane 12remaining intact has the advantage of avoiding contamination of thesample. For example the membrane 12 remains intact while being shaken bya cellular homogenizer wherein particulate material 20 is agitatedwithin the cartridge 10 and contacts the membrane 12.

Integrated System

Another aspect of the invention is a system for isolatingsingle-stranded DNA from a sample containing cells, comprising: acontainer 10 having an entrance port 16 and an exit port 18, a membrane12 disposed within the container 10 between the entrance 16 port and theexit port 18, and a plurality of channels 14 located between themembrane 12 and the exit port 18; and an amplification and separationsystem comprising an amplification container 50, a thermal cycler, and amagnetic substrate 52.

In some embodiments the system integrates a device for isolating DNAfrom a sample containing cells, as described hereinabove, with a systemfor amplification and separation to amplify and isolate single-strandedDNA.

The system also may include pumps, valves, channels, tubes, fittings,syringes, chambers, containers, and/or vacuum sources to transfer asample containing cells into and out of the cartridge 10 and into andout of an amplification container 50. For example, the system isconfigured to flow an initial sample containing cells suspended in aliquid into the cartridge 10 and remove the liquid from the cells bypassing the liquid through the membrane 12 and channels 14 and out theexit port 18 of the cartridge 10 leaving cells (and non-DNA cellularmaterial) trapped on the membrane 12. In some embodiments the cellstrapped on the membrane within the cartridge are re-suspended in aliquid, for example, a buffer. In some embodiments the system isconfigured to re-suspend the cells trapped on the membrane in a smallvolume of liquid to increase the concentration of DNA per volume andimprove downstream applications.

In one embodiment the system further comprises a vacuum configured topull a liquid sample containing cells through the cartridge 10 whereinthe cells are trapped on the membrane 12 and the liquid is removed fromthe cartridge 10. The system may also include a syringe containing aliquid sample configured to be attached to the entrance port 16.

In some embodiments the cartridge is sized and configured to fit withina cellular homogenizer capable of shaking the cartridge 10 to agitatethe particulate material 20 (e.g., microbeads) within the lysis chamber22. For example, the cellular homogenizer can shake the cartridge atspeeds up to about 5000 rpm.

In some embodiments the amplification and separation system comprisesthe amplification container 50 for amplifying DNA via PCR. In someembodiments the amplification container 50 is sealed to hold in moistureduring PCR. For example, the amplification container 50 includes a waxseal at a lid of the container to hold in moisture during PCR, whereinthe wax seal melts to seal the lid of the amplification container 50. Insome embodiments the amplification container 50 is a plastic tube, forexample, a microcentrifuge tube. In one embodiment, the amplificationcontainer 50 can hold a volume of liquid of up to about 1 or 2 mL.

A thermal cycler may be included to control the temperature within theamplification container 50. In some embodiments the thermal cycler isprogrammed to subject the container to predetermined PCR thermal cycles.For example the thermal cycler is programmed to subject the container toa plurality of thermal cycles for PCR followed by a plurality of cyclesfor asymmetric PCR.

In some embodiments the amplification container 50 is loaded with PCRreagents. For example, the amplification container is loaded withprimers, enzymes (DNA polymerase), and nucleotides and/or other PCRcomponents and reagents known in the art. For example the amplificationcontainer is loaded with reagents such that the reagents are optimizedto undergo PCR followed by asymmetric PCR. In some embodimentsasymmetric PCR is used to generate single-stranded DNA. Morespecifically, asymmetric PCR may be used to generate single-stranded DNAof a specific length or within a specific range of lengths, for examplessDNA of from about 10 to about 200 bases or, for example, less than 100bases.

In another embodiment, the amplification container 50 contains magneticsubstrate 52, such as magnetic beads. In some embodiments the magneticbeads are silica-based beads. In one example the magnetic beads areparamagnetic beads. In some embodiments the magnetic substrate 52 is aplurality of substantially spherical beads of about 1 micron to about100 microns, for example about 10 microns in diameter.

In some embodiments the magnetic substrate 52 includes a coating that iscapable of attracting double-stranded DNA but does not substantiallyattract single-stranded DNA. In some embodiments the coating has abinding affinity for double-stranded DNA that is greater than itsbinding affinity for single-stranded DNA. In some embodiments thecoating decreases the time for elongating ssDNA during asymmetric PCR.In some embodiments the coating comprises a carboxylic acid linker. Insome embodiments the coating comprises N-hydroxysuccinamide (NHS).

In some embodiments the amplification and separation system furtherincludes a magnet configured to apply a magnetic field to the magneticsubstrate. For example, the magnet is positioned underneath theamplification container. The magnet may be a paramagnet.

In some embodiments the systems are small, portable, and inexpensiverelative to known systems.

III. Methods

Another aspect of the invention is a method of isolating DNA from asample, comprising providing a sample containing cells (i.e., anybiological sample that includes cells); transferring the sample into acontainer having a membrane and a plurality of channels disposedtherein; lysing the cells to release DNA and non-DNA cellular material;passing DNA through the membrane; and passing the DNA through thechannels.

In some embodiments, the sample containing cells is a liquid sample. Forexample the sample comprises blood, urine, water, or exudate.

The cells in the sample may be any cells that include DNA, for example,bacteria. Exemplary cells include E. coli, Listeria, Brucella, MRSA andEnterococcus. In some embodiments the DNA is isolated from a blood orurine sample from a patient being tested for a disease, for example,lymphatic filariasis or dengue fever.

In one embodiment, the sample is transferred into the cartridge 10 (e.g.as described above) using a vacuum. In another embodiment, the sample istransferred into the cartridge using a pump.

More specifically, for example, a liquid sample is drawn or pushed intothe cartridge 10 and the cells are trapped on membrane 12, while theliquid flows through the membrane 12 and channels 14 and exits thecartridge 10 and is collected as waste.

In some embodiments a liquid, e.g. a buffer, is pushed or drawn into thecartridge 10 and the cells trapped on the membrane 12 are re-suspendedin the liquid. The liquid may be selected from water, a buffer such as alysis buffer, saline buffer, mild ionic buffer, or strong ionic buffer.The buffer can be selected on the basis of its interaction with cells(e.g., lysis buffer, isotonic buffer), its interaction with DNA (e.g.,hybridization buffer), its effect on the interaction of cells and DNAwith the membrane (e.g., an elution buffer) or its suitability fordownstream applications such as amplification or detection methods (e.g.PCR buffer, hybridization buffer, high or low conductivity buffer). Inone embodiment, the buffer is phosphate-buffered saline (PBS).

Another step of the inventive method may include re-suspending the DNAonly once while it pushed through the cartridge 10 to be isolated. Forexample, the DNA does not undergo multiple washing and elution steps. Insome embodiments the DNA is suspended in a small volume of buffer (e.g.less than 100 mL in order to concentrate the DNA and allow for moreefficient downstream functions).

Using the present method the cells are trapped within lysis chamber 22,a region between the entrance port 16 and the membrane 12. In someembodiments the cells are lysed within the lysis chamber 22. In otherembodiments, the cells are lysed prior to being transferred to thecartridge 10.

The cells may be lysed by chemical, mechanical, thermal, or electricalmeans. For example, the cells are lysed using a lysis buffer such astris-HCl, EDTA, EGTA, SDS, deoxycholate, triton, NP-40 and/or a buffercontaining NaCl or the cells may be lysed by heating the cells. Thecells may also be lysed by mechanical means such as bead beating, shearforces, gas bubble agitation, or sonication.

In some embodiments the cells are mechanically lysed using particulatematerial 20 (e.g. beads or microbeads), contained within lysis chamber22. For example a cellular homogenizer shakes cartridge 10 to agitateparticulate material 20 and the cells within the cartridge 10. Thepurpose of the particulate material 20 is to rupture the cell membranesor walls of the cells and release DNA. In one embodiment, the beads areglass microbeads or stainless steel microbeads. During use, the cellularhomogenizer shakes cartridge 10, e.g., at a rate of about 3000 rpm. Thenon-DNA cellular material may be substantially destroyed leavingbiohazard free waste. In some embodiments, the cellular homogenizershakes cartridge 10 for about 60 to about 300 s. The non-DNA cellularmaterial is substantially destroyed when, for example 50%, 60%, 70%,80%, 90%, 95%, or 99% or more of the non-DNA cellular material isdestroyed.

In some embodiments the DNA released from the cells flows throughmembrane 12. For example, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or moreof the DNA released from the cells flows through membrane 12. In someembodiments the non-DNA cellular material does not flow through membrane12. For example 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of thenon-DNA cellular material does not flow through membrane 12.

In some embodiments the released DNA that flows through membrane 12subsequently flows through channels 14. For example 50%, 60%, 70%, 80%,90%, 95%, or 99% or more of the DNA that flows through membrane 12 willthen flow through channels 14. In some embodiments the yield of DNAisolated from the sample is greater than 50%, 60%, 70%, 80%, 90%, 95%,or 99%.

In some embodiments the DNA that flows through channels 14 flows outexit port 18 of cartridge 10. The DNA may be suspended in liquid, e.g.buffer, and the suspension is collected. For example, the suspension iscollected and transferred to a downstream application, such anamplification or detection function. In some embodiments the DNA ispushed with a dry syringe (air) into a separate container for downstreamapplications.

Another aspect of the invention is a method of amplifying and isolatingsingle-stranded DNA from a sample containing DNA, comprising providing asample containing DNA; amplifying the DNA via a polymerase chainreaction (PCR), wherein the PCR includes asymmetric PCR, and wherein thePCR produces a mixture comprising double-stranded DNA andsingle-stranded DNA; providing a magnetic substrate 52, wherein themagnetic substrate 52 has an affinity for binding double-stranded DNA;applying a magnetic field to the magnetic substrate 52; and isolatingthe unbound single-stranded DNA from the sample.

Another aspect of the invention provides combining the method ofisolating DNA from a sample containing cells, as described herein, withthe method of amplifying and isolating single-stranded DNA from a samplecontaining DNA, as described herein, such that an initial samplecontaining cells is processed to produce a sample comprising isolatedsingle-stranded DNA.

In some embodiments the amplification comprises traditional PCR followedby asymmetric PCR. In some embodiments the traditional PCR followed byasymmetric PCR produces a mixture of double-stranded DNA andsingle-stranded DNA. This protocol may produce a portion of the ssDNAwithin a specified range of lengths. For example, a portion of the ssDNAis approximately 50 to 200 bases in length or a portion of the ssDNA isapproximately 100 bases in length. For example the concentration ofssDNA within a specified range of lengths is greater than about 100 nM.For example the concentration of ssDNA with a length of 50 to 200 basesor a length of approximately 100 bases, is greater than about 100 nM.

In some embodiments, the mixture of dsDNA and ssDNA undergoes aseparation procedure to separate the ssDNA from the dsDNA. In oneembodiment the dsDNA is bound to magnetic substrate 52, for example, aplurality of magnetic beads. In this embodiment, the dsDNA is attractedto a coating on the magnetic beads and binds to the coating on themagnetic beads. The magnetic substrate 52 may comprise a carboxylic acidlinker. In some embodiments the magnetic substrate also functions toelongate ssDNA during asymmetric PCR.

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the dsDNA in the mixture is bound to the magnetic substrate 52 and,respectively, less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the dsDNAin the mixture is not bound to the magnetic substrate 52. In otherembodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thessDNA in the mixture is recovered from the mixture. ssDNA isolated bythis process may be used in downstream applications, for example, in adetection assay based on hybridization of the ssDNA with a specific DNAprobe. In some embodiments, the unbound ssDNA is isolated from themixture. For example, liquid is slowly drawn from the sample, while themagnetic beads bound to the dsDNA remain held by the magnet. In someembodiments, the isolated unbound ssDNA as compared to the unbound andbound dsDNA comprises a ratio of greater than 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 99:1 of unbound ssDNA to unbound/bound dsDNA. In oneexample, this ratio is 100:1 and in another example 1000:1 of unboundssDNA to unbound/bound dsDNA.

In some embodiments the methods can be performed in a reduced timecompared to known methods, and/or with reduced technician time requiredto carry out the process. In some embodiments the methods do not requiremultiple steps, such as washing and elution steps. In some embodimentsthe methods result in a high yield of DNA and/or single-stranded DNA.

IV. EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example No. 1 Process Protocol

With reference to FIGS. 6A, 6B, 6C, and 6D an example protocol isdescribed. A vacuum bottle (or “waste bottle”) is set up where one endof a vacuum tube is inserted into a cap hole in the vacuum bottle andthe other end of the vacuum tube is inserted into a vacuum source. Aplastic fitting is inserted into a second cap hole. See, FIGS.6A(i)-6A(iii).

A cartridge is attached to the fitting on the second cap hole of thevacuum bottle. A funnel is attached to the top of the cartridge usingthe luer lock fittings. See, FIGS. 6A(iv)-6A(v).

A sample containing cells with a volume of about 5 to 100 mL is obtainedand transferred into the funnel. The vacuum pump is activated and fluidis drawn from the funnel, through cartridge 10, and into the vacuumbottle. The cells in the sample are trapped on membrane 12 (not shown)within cartridge 10. See, FIGS. 6A(vi)-6A(vii).

The funnel is removed from cartridge 10 and cartridge 10 is removed fromthe fitting on the cap of the vacuum bottle. A buffer syringe with PBSbuffer is attached to the top of cartridge 10. See, FIGS.6A(viii)-6A(x). The buffer syringe is attached to cartridge 10 andbuffer is slowly pushed into cartridge 10 at a flow rate of about 0.5 to4 mL/s. The volume of buffer pushed into cartridge 10 is from about 10mL to about 30 mL. Cartridge 10 is closed with caps on entrance port 16and exit port 18. The process time for the steps above is from about 15to about 120 seconds. See, FIGS. 6B(i)-6B(ii).

Cartridge 10 is placed inside a cellular homogenizer. See, FIG.6B(iii).The cellular homogenizer is activated and shakes cartridge 10.See, FIG. 6B(iv). The cellular homogenizer operates for 60 to 300seconds at a speed of about 1000 to 3500 rpm. The cellular homogenizeris then deactivated and cartridge 10 is removed.

An empty (air) syringe is attached to entrance port 16 of cartridge 10and a needle is attached to exit port 18. See, FIG. 6B(v) and FIG.6C(i). The syringe is slowly depressed to push the liquid insidecartridge 10 through membrane 12 and channels 14 and into a sample vial.See FIG. 6C(ii). The sample vial is prefilled with magnetic substrate 52and PCR reagents. Forms of magnetic substrate 52 are commerciallyavailable, e.g., magnetic beads such as PureProteome NHS FlexibindMagnetic Beads, Millipore product number LSKMAGNXXMAN. The PCR reagentsare commercially available, GE Healthcare Illustra PuRe Taq Ready-to-GoPCR. Primers are specially designed for the desired assay. Commerciallyavailable primers may be made by IDTDNA.

The sample vial is loaded into a thermal cycler (optimizer). See FIG.6C(iii). The DNA in the sample vial undergoes PCR followed by asymmetricPCR for about 20-45 minutes in the optimizer. PCR includes a ramp up to90-98 degrees Centigrade for 2 minutes. 18-30 total cycles of PCR andasymmetric PCR amplify the DNA. The cycles include a hot step at 90-98degrees Centigrade for 10-20 seconds, a cool step at 55-65 degreesCentigrade for 15-25 seconds, and a copy step at 70-75 degreesCentigrade for 25-35 seconds. The sample undergoes a final cool downstep at 70-75 degrees Centigrade for 3 min.

The sample is removed from the optimizer and placed in a sample vialholder. See FIG. 6C(iv). The sample vial holder is equipped with amagnet to attract magnetic substrate 52 (bound to dsDNA) and holdmagnetic substrate 52 at the bottom of the sample vial. The liquid(containing ssDNA) is then withdrawn from the sample vial and sent todownstream applications, such as a DNA biochip detection assay.6D(i)-6D(iv).

Example No. 2 Method of Manufacture

With reference to FIG. 7, an example of a method of manufacture ofcartridge 10 is described. Cartridge 10 is assembled from multiplecomponents (from top to bottom): a luer cap 31, top piece 32 (“top SPC”or “top sample prep cartridge”), particulate material 20 (glass beads),membrane 12, bottom piece 34 (“bottom SPC” or “bottom sample prepcartridge”), and a luer plug 33.

Luer cap 31 and luer plug 33 are commercially available and configuredto attach to standard luer fittings at entrance port 16 and exit port 18of the top SPC 32 and bottom SPC 34. Luer cap 31 and Luer plug 33 holdcontents within cartridge 10. 0.3 grams of particulate material 20(glass beads) are weighed and loaded in cartridge 10.

Membrane 12 is held in place by the connection between top SPC 32 andbottom SPC 34.

Top SPC 32 and bottom SPC 34 are made from plastic. Cartridge 10 isdesigned to accept up to 100 mL of liquid. Top SPC 32 and bottom SPC 34are sonic-welded together and fix membrane 12 in place. Top SPC 32 andbottom SPC 34 have an entrance port 16 and an exit port 18,respectively, with standard luer lock fittings. Top SPC 32 and bottomSPC 34 are made using three dimensional printing. Bottom SPC 34 includescontains channels 14 in the form of a matrix. The channel matrix is madeusing three dimensional printing for bottom SPC 34 configured to formbottom SPC with a plurality of channels 14.

For assembly, membrane 12 is inserted into bottom SPC 34, top SPC 32 isinserted into bottom SPC 34, the components are sonic welded together,luer plug 33 is attached to bottom SPC 34, particulate material 20 isinserted into top SPC 32 through the entrance port 16, and luer cap 31is attached.

Example No. 3 Cartridge Embodiment

With reference to FIGS. 8A-C and 9A-B an example of an embodiment ofcartridge 10 is shown. FIGS. 8A-C and 9A-B show the architecture of anexample of top SPC 32 and bottom SPC 34, respectively, as describedherein. Top SPC 32 and bottom SPC 34 may be sonic welded together toform cartridge 10.

With reference to FIGS. 8A-C, top SPC 32 has a cylindrical shape with atop surface, an entrance port 16 having standard luer lock fittings 17extending from the center of the top surface, and side wall extendingdownward from the edge of the top surface.

With reference to FIGS. 9A-B, bottom SPC 34 has a cylindrical shape witha bottom surface, an exit port 18 having standard luer lock fittings 19extending from the center of the bottom surface and side walls extendingupward from the edge of the top surface. Further a plurality of channels14 are formed within bottom SPC 34. Channels 14 form a pattern extendingradially outward from the center of bottom SPC 34.

Example No. 4 E. coli Test

A variety of ground beef samples were tested using the F Cubed NESDEP IU(FIG. 5) and benchmark plate cultures. The pathogen E. coli 0157:H7 wasinoculated into ground beef samples. The sample concentration was 10²cells per mL. The sample was processed as described in the processprotocol of Example No. 1. The isolated ssDNA sample then underwentdetection using an electrochemical detection assay. An example ofimpedance data generated from an electrochemical detection assay isshown in FIG. 10. Sensitivity, specificity, negative predictive value,and positive predictive value were all 100%. Results are shown in Table1.

TABLE 1 E. coli test results SITE SAMPLE F3 ASSAY PLATE SAMPLE ASSAY ID¹NUMBER² RESULT³ RESULT⁴ TYPE⁵ PROCESS⁶ NOTES A 1 Positive Yes 75% Q 1.5CFU Index = 1.11 A 2 Positive Yes 75% Q 5.0 CFU Index = 1.20 A 3Positive Yes 75% Q 15.0 CFU Index = 1.35 A 4 Positive Yes 60% Q 1.5 CFUIndex = 1.11 A 5 Positive Yes 60% Q 6.0 CFU Index = 1.03 A 6 PositiveYes 60% Q 15.0 CFU Index = 1.20 A 7 Negative No 75% Q 6.0 CFU Index =0.01 A 8 Positive Yes 75% Q 15.0 CFU Index = 1.35 A 9 Negative No 60% Q1.5CFU Index = 0.87 A 10 Positive Yes 60% Q 6.0 CFU Index = 1.13 LEGEND¹(A) F3 Labs/(B) Off-Site/(C) Undefined ²F3 Number Sequence ³F3 NESDEPIU System ⁴Plate Culture With Manual Smear And Incubate ⁵SamplesObtained From Martin'S Supermarkets (60% CB Or 75% CB) ⁶(Q) QualitativeF3 NESDEP/(F) Quantitative F3 NESDEP

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A device for isolating DNA from a samplecontaining cells, comprising a cartridge having an entrance port and anexit port; a membrane disposed between the entrance port and the exitport; and a plurality of channels between the membrane and the exitport.
 2. The device of claim 1, wherein the channels are adjacent to themembrane.
 3. The device of claim 1, wherein each of the channels has achannel entrance, a channel exit, and a channel wall, wherein thechannel entrance is adjacent to the membrane.
 4. The device of claim 3,wherein the channel entrance has a length and width, wherein the lengthis about 0.1 mm to about 100 mm and the width is about 0.1 mm to about10 mm.
 5. The device of claim 1, having from about 2 to about 1000channels.
 6. The device of claim 1, wherein the channels are arranged ina matrix.
 7. The device of claim 6, wherein the channels are adjacent toeach other.
 8. The device of claim 6, wherein the channels are spacedapart from each other.
 9. The device of claim 8, further comprising asolid barrier between each channel entrance.
 10. The device of claim 1,wherein the membrane is configured to be permeable to DNA butsubstantially impermeable to cells.
 11. The device of claim 1, whereinthe membrane is configured to be permeable to DNA but substantiallyimpermeable to non-DNA cellular material.
 12. The device of claim 1,wherein the membrane comprises a cellulose material.
 13. The device ofclaim 1, wherein the membrane has pores.
 14. The device of claim 13,wherein the pores are from about 10 nm to 1 micron in diameter.
 15. Thedevice of claim 1, further comprising a plurality of particulatematerial located between the membrane and the entrance port.
 16. Thedevice of claim 15, wherein the particulate material comprises glass orstainless steel.
 17. The device of claim 15, wherein the particulatematerial comprises a plurality of microbeads.
 18. The device of claim17, wherein the microbeads have a diameter of from about 1 micron toabout 1000 microns.
 19. The device of claim 1, wherein the entrance portand exit port comprise standard luer lock fittings.
 20. The device ofclaim 1, wherein the cartridge includes a bottom piece that supports themembrane and the plurality of channels are formed from cavities in thebottom piece of the cartridge.
 21. A system for isolating DNA from asample containing cells comprising the device of claim 1, and a vacuumto pull fluid into the cartridge.
 22. The system of claim 21, furthercomprising a cellular homogenizer to shake the cartridge.
 23. The systemof claim 22, wherein the cellular homogenizer is capable of shaking thecartridge at a rate of up to 5000 rpm.
 24. The system of claim 22,further comprising a thermal cycler configured to subject the DNA tothermal cycling for a polymerase chain reaction (PCR).
 25. The system ofclaim 24, further comprising a magnetic substrate wherein the magneticsubstrate is a coating with a binding affinity for double-stranded DNA.26. The system of claim 25, further comprising a magnet configured toapply a magnetic field to the magnetic substrate.
 27. The system ofclaim 25, wherein the magnetic substrate is a plurality of magneticbeads.
 28. A method of processing DNA from a sample, comprisingproviding a sample containing cells having DNA; transferring the sampleinto a cartridge having a membrane and a plurality of channels disposedtherein; lysing the cells to release the DNA and non-DNA cellularmaterial; passing the DNA through the membrane; and passing the DNAthrough the channels; wherein the DNA is isolated from the sample. 29.The method of claim 28, wherein the sample comprises blood, exudate,water, or food.
 30. The method of claim 28, wherein the transferringcomprises using a vacuum to transfer the sample into the cartridge. 31.The method of claim 28, wherein the lysing comprises mechanical lysing.32. The method of claim 31, wherein the mechanical lysing comprisesagitating a plurality of particulate material disposed within thecartridge.
 33. The method of claim 32, wherein the mechanical lysingsubstantially destroys the non-DNA cellular material.
 34. The method ofclaim 28, wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe non-DNA cellular material in the sample does not pass through themembrane.
 35. The method of claim 28, wherein greater than 50%, 60%,70%, 80%, 90%, 95%, or 99% of the DNA in the sample is passed throughthe membrane and the channels.
 36. The method of claim 28, furthercomprising transferring the isolated DNA to a DNA amplification chamberand amplifying the isolated DNA by a polymerase chain reaction (PCR),wherein the PCR is asymmetric, to produce double-stranded DNA andsingle-stranded DNA.
 37. The method of claim 36, wherein a portion ofthe single-stranded DNA is within a specified range of lengths.
 38. Themethod of claim 36, further comprising binding the double-stranded DNAto a magnetic substrate and applying a magnetic field to the substrate,wherein the magnetic field attracts the substrate bound to thedouble-stranded DNA, and isolating the single-stranded DNA.
 39. A methodof amplifying and isolating single-stranded DNA from a sample containingDNA, comprising providing a sample containing DNA; amplifying the DNAvia a polymerase chain reaction (PCR), wherein the PCR is asymmetricPCR, to produce a mixture of double-stranded DNA and single-strandedDNA; providing a magnetic substrate having an affinity for binding thedouble-stranded DNA; binding the magnetic substrate to thedouble-stranded DNA; applying a magnetic field to the magneticsubstrate, wherein the magnetic field attracts the magnetic substratebound to the double-stranded DNA; and isolating the single-stranded DNAfrom the mixture.
 40. The method of claim 39, wherein PCR comprisestraditional PCR followed by asymmetric PCR.
 41. The method of claim 39,wherein a portion of the single-stranded DNA is within a specified rangeof lengths.
 42. The method of claim 41, wherein at least 50%, 60%, 70%,80%, 90%, 95%, or 99% of the single-stranded DNA are within a specifiedrange of lengths.
 43. The method of claim 41, wherein a portion of thesingle-stranded DNA is no more than about 200 bases in length.
 44. Themethod of claim 43, wherein a portion of the single-stranded DNA is nomore than about 100 bases in length.
 45. The method of claim 39, furthercomprising an amplification container, placing the sample in theamplification container, and sealing the amplification container with awax seal.
 46. The method of claim 39, wherein the magnetic substrate hasa coating with an affinity for binding double-stranded DNA.
 47. Themethod of claim 46, wherein the coating that does not substantiallyattract single-stranded DNA.
 48. The method of claim 39, wherein themagnetic substrate is a paramagnetic substrate.
 49. The method of claim39, wherein the magnetic substrate is a plurality of magnetic beads. 50.The method of claim 39, wherein greater than 50%, 60%, 70%, 80%, 90%,95%, or 99% of the double-stranded DNA in the mixture is bound to themagnetic substrate and, respectively, less than 50%, 40%, 30%, 20%, 10%,5%, or 1% of the double-stranded DNA in the mixture is not bound to themagnetic substrate.
 51. The method of claim 39, wherein greater than50%, 60%, 70%, 80%, 90%, 95%, or 99% of the single-stranded DNA in themixture is isolated.
 52. The method of claim 39, wherein the isolatedunbound single-stranded DNA as compared to the unbound and bounddouble-stranded DNA comprises a ratio of greater than 50:50, 60:40,70:30, 80:20, 90:10, 95:5, 99:1, 100:1 or 1000:1.